Estimation of Total Factor Productivity Growth In

Pak. J. Agri. Sci., Vol. 56(4), 943-951; 2019 ISSN (Print) 0552-9034, ISSN (Online) 2076-0906 DOI:10.21162/PAKJAS/19.3948 http://www.pakjas.com.pk DIVERSITY PROFILE OF PROTISTS FLAGELLATES ISOLATED FROM HINDGUT OF Heterotermes indicola WASMANN (BLATTODEA: RHINOTERMITIDAE) IN PAKISTAN Asma Ashraf1, Naveeda Akhtar Qureshi1,*, Muhammad Haseeb1, Muhammad Qasim Hayat2, Muhammad Afzal1 and Saeed-ul-Hassan Khan1 1Department of Animal Sciences, Faculty of Biological Science, Quaid-i-Azam University Islamabad 45320, Pakistan; 2Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST), Islamabad, Pakistan *Corresponding author’s e-mail: [email protected] Termites cause a serious menace to wood structures all over the world. They rely mostly on the entozoic fauna for the digestion of cellulosic materials. The present study is based upon the diversity of flagellates protists isolated from the gut of a lower termite, Heterotermes indicola, belonging to three genera i.e. Holomastigotes (H. campanula, H. annandalei and H. metchnikowi), Holomastigotoides (H. hemigynum, H. hartmanni, H. kempi, H. koidzumi and H. metchnikowi) and Pseudotrichonympha (P. grassii). The largest and most abundant species Pseudotrichonympha grassii was identified by molecular studies using the SSU rRNA gene, confirmed by phylogenetic analysis and compared with that of the P. grassii isolates reported from other parts of the world. The results showed that the P. grassii observed in our study was phylogenetically most closely related to the Japanese P. grassii isolate. The biodiversity of the entozoic flagellates is important in targeting for biological control of termites as well as for isolation and culturing of flagellates to produce cellulases, an important industrial enzyme. Keywords: Biodiversity, Heterotermes indicola, entozoic flagellates, Holomastigotes, Holomastigotoides, Pseudotrichonympha. INTRODUCTION endomicrobes found in the hindgut of termites play a major role in cellulose digestion and manufacture cellulases that Heterotermes indicola (Blattodea: Rhinotermitidae) is a break down cellulose into acetate, propionate and butyrate. lower termite which harbor entozoic flagellates for cellulose They also fix nitrogen that gets incorporated into the termite’s digestion thus causing considerable damage to forests and tissues, excrement and secretions (Mathew et al., 2012). wooden materials throughout the world except Antarctica. Recycling of uric acid by the termite gut bacteria is another About 434 flagellate’s species have been identified in the source of nitrogen (Thong-On et al., 2012). With the aid of hindgut of different termite species which belong to one of the these endomicrobes, such small insects (termites) cause three orders: Trichomonadida, Hypermastigida and damage worth billions to woods and wooden materials. The Oxymonadida (Inoue et al., 2000). During the last two information pertaining to protozoa, found in the termites as decades termite gut flagellates have been classified using symbionts, will be helpful towards designing strategies for the molecular methods, which are considered to be good tools for production of cellulases and control of lower termites via the species identification and for defining evolutionary inferences development of drugs targeting these protozoa. (Yang and Rannala, 2012). Saldarriaga et al. (2011) described Owing to their importance in the termite hindgut, information the morphology and phylogeny of two Pseudotrichonympha about these endomicrobes and their diversity is extremely species i.e. P. hertwigi from Coptotermes testaceus and P. important. The present work was aimed to investigate the paulistana from Heterotermes tenuis. Phylogenetic analysis diversity of flagellated protozoa with a special focus on P. of fifteen species of Pseudotrichonympha was described by grassii, the largest and the most abundant protozoan found in Noda et al. (2007) from different termite species. However, the hindgut of H. indicola. Moreover, to control the he suggested that only one species of Pseudotrichonympha is population rate of termites and to protect the huge wood found in a termite species at one time. Thus the evolutionary damage and economic loss, it is necessary to eliminate the history of Pseudotrichonympha is important not only due to population of gut flagellates which play an important role in its host specificity but as a host of bacterial endosymbionts, cellulose digestion of termites and made symbiotic which account for more than two third of the total bacterial relationship with each other. population in the termite gut (Noda et al., 2007).The Ashraf, Qureshi, Haseeb, Hayat, Afzal & Khan MATERIALS AND METHODS SSU rRNA gene. The PCR was performed on five cells. An initial PCR was done by using set of forward and reverse Study area and Collection of termites: Workers of H. primers (Table 1). The reaction mixture consisted of template indicola were collected from the metropolitan areas of cells, dNTP mix (0.2 mM), primers (1µM each), PCR Buffer Islamabad (33.729388°N, 73.093146°W) (Fig. 1) Pakistan (1X), Ex-Taq DNA polymerase (2.5U) and MgCl2 (1.5mM). from March to May 2017. Termites were kept in Petri dishes Amplification was consisted of 35 cycles of 1 min at 92°C, 1 at 30°C and fed on moistened blotting paper for a week prior min at 50°C and1.5 min at 72°C. Agarose gel electrophoresis to the experiment and identified by using key (Akhtar, 1983). of the reaction revealed no bands. Therefore, using the first This helped in clearing debris and wood particles from reaction contents as a template, a nested PCR was performed. flagellates for distinct morphology. 25µl of PCR products was added with the same constituents as used for primary PCR by using the second set of primers for nested PCR (Table 1). The pieces of gel containing the DNA band were excised from the gel and subjected to DNA purification using the Gene JET Gel Extraction Kit (Cat No. K0691, MBI Fermentas). The pGEM-T-Easy vector (Cat No. A1360, Promega) was used to clone the purified products and transformed into DH5α E. coli cells. This was followed by plasmid DNA extraction and DNA sequencing of the cloned PCR product. Phylogenetic analysis: For the phylogenetic investigation, we analyzed our indigenous P. grassii taxon and compared it with that of isolated from the other localities of the world. The details are given in Table 4. The sequence alignment and data matrix construction were done in the software Geneious® version 6.1 (Biomatters Ltd., New Zealand). Neighbor joining Figure 1. Termites collection sites from Islamabad, analysis was performed using the DNA evolution model Pakistan. (Tamura and Nei, 1993) in the Geneious build algorithm. Wagner parsimony (Farris, 1970) and Maximum likelihood Light microscopy: The hindgut contents of termites were (Guindon and Gascuel, 2003) analysis were carried out using opened in a drop of 0.2% normal saline and tinged with gram PAUP (Swofford, 2002) Geneious plugin. Bayesian analysis iodine solution. Flagellates were observed by using Optika was done using MrBayes (Huelsenbeck and Ronquist, 2001) trinocular digital microscope (Optika B-350, Italy). Images Geneious plugin. Finally, based on the above analysis a and videos were captured in real time with a 12 megapixels consensus evolutionary tree was generated. digital CCD camera on a trinocular Optika microscope. Single cell isolation of P. grassii: The gut of termite workers RESULTS was opened in a 5μl of filter-sterilized buffer (0.1 M NaC1, 10mM Na3PO4, pH 6.9) in the cavity slide. From this mixture Flagellates biodiversity: The protozoan fauna isolated from of gut contents and the buffer, 1ml was taken on another hindgut of H. indicola were categorized according to Adl et cavity slide and diluted further by adding 4ml of the buffer. al. (2005). We identified nine species of flagellates belonged This dilution was repeated four times, 1ml of the final diluted to three genera Holomastigotes, Holomastigotoides and contents was used for observations. The flagellates were Pseudotrichonympha. examined under a digital biological microscope. Individual P. Taxonomic classification of flagellates isolated from H. grassii cells, identified on the basis of morphology, were indicola: picked with a micropipette and collected in PCR tubes. Class Mastigophora (Diesing): Member of class PCR amplification and sequencing: The cells were directly Mastigophora usually have one to several flagella, the nucleus used as a template in a PCR to amplify their nearly full length is vesicular with endosomes. Table 1. Set of primers used for primary and nested PCR. Primer Name Sequence Reference Primary PCR Eukl8 5’-TGAGGATCCMGGTTGATYCTGCC-3’ Ohkuma et al. (2000) Euk1627 5’-CCGAAGCTTACGGGCGGTGTGTRC-3‘ Nested PCR Har-F 5’-GCGCTACCTGGTTGATCCTGCC-3’ Harper et al. (2009) Har-R 5’-TGATCCTTCTGCAGGTTCACCTAC-3’ 944 Profile of protists flagellates Sub Class Zoomastigia: These may be free living or parasitic deeply stained spiral bands which cover most of the body; and their body lacks chromatophores. Ensysment is usually flagella arise from basal granules; bulge may or may not be common. present at posterior end of body; a long axostyle present; body Order Hypermastigida (Grassii and Foa): Flagellates covered with periplast. belonging to the order Hypermastigida have numerous KEY TO SPECIES flagella and complex cytoplasmic organization. KEY TO FAMILY Holomastigotes campanula: Diagnosis: Campanula shaped body; broadly round at the KEY TO

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